Sacbrood viruses and select Lake Sinai virus variants dominated Apis mellifera colonies symptomatic for European foulbrood

ABSTRACT European foulbrood (EFB) is a prevalent disease in the European honey bee (Apis mellifera) in the United States, which can lead to colony decline and collapse. The bacterial components of EFB are well-studied, but the diversity of viral infections within infected colonies has not been explored. In this study, we use meta-transcriptomics sequencing of 12 honey bee hives, symptomatic (+, n = 6) and asymptomatic (–, n = 6) for EFB, to investigate viral infection associated with the disease. We assembled 41 viral genomes, belonging to three families (Iflaviridae, Dicistroviridae, and Sinhaliviridae), all previously reported in honey bees, including Lake Sinai virus, deformed wing virus, sacbrood virus, Black queen cell virus, and Israeli acute paralysis virus. In colonies with severe EFB, we observed a higher occurrence of viral genomes (34 genomes) in contrast to fewer recovered from healthy colonies (seven genomes) and a complete absence of Dicistroviridae genomes.We observed specific Lake Sinai virus clades associated exclusively with EFB + or EFB – colonies, in addition to EFB-afflicted colonies that exhibited an increase in relative abundance of sacbrood viruses. Multivariate analyses highlighted that a combination of site and EFB disease status influenced RNA virome composition, while EFB status alone did not significantly impact it, presenting a challenge for comparisons between colonies kept in different yards. These findings contribute to the understanding of viral dynamics in honey bee colonies compromised by EFB and underscore the need for future investigations to consider viral composition when investigating EFB. IMPORTANCE This study on the viromes of honey bee colonies affected by European foulbrood (EFB) sheds light on the dynamics of viral populations in bee colonies in the context of a prevalent bacterial brood disease. The identification of distinct Lake Sinai virus and sacbrood virus clades associated with colonies affected by severe EFB suggests a potential connection between viral composition and disease status, emphasizing the need for further investigation into the role of viruses during EFB infection. The observed increase in sacbrood viruses during EFB infection suggests a potential viral dysbiosis, with potential implications for honey bee brood health. These findings contribute valuable insights related to beekeeping practices, offering a foundation for future research aimed at understanding and mitigating the impact of bacterial and viral infection in commercial honey bee operations and the management of EFB.

significantly impacted bee health in recent years.Data from 2022 provided by the Bee Informed Partnership show that US commercial beekeepers experienced 46.1% loss in their colonies from 2019 to 2020 (2).Many factors can contribute to colony loss, such as the physiological condition of adult bees, pathogen loads, and pesticide levels (3,4).European foulbrood, commonly referred to as EFB, is a highly prevalent bacterial disease affecting honey bee larvae in the United States, with Melissococcus plutonius identified as the primary pathogen based on genomic studies, field observations, and laboratory testing (5,6).Two studies have recently focused on EFB in the United States, and both have noted high rates of disease and risk to colony growth, particularly after blueberry field pollination (7,8).Honey bees infected with EFB can exhibit high mortality; however, disease monitoring is complicated by M. plutonius being present in both symptomatic and asymptomatic colonies (9,10).Currently, the only approved treatment in the United States for EFB is the antibiotic oxytetracycline, for which antimicrobial-resistant strains have been detected in North American honey bee colonies (11).Not only are better treatments urgently needed, but improved fundamental knowledge of the disease and its progression, in addition to the onset of secondary infections, is also necessary for disease management.
Stress factors associated with EFB may leave colonies susceptible to secondary infections.The monitoring and understanding of the range of secondary pathogens that can complicate the management of the disease and treatments can be as important as diagnosing the primary infection.EFB secondary bacterial infective agents include Enterococcus faecalis and Paenibacillus alvei and are well-described (6,7,9).Secondary viral infections have been largely overlooked, and comparisons between EFB symp tomatic (+) and asymptomatic (-) colonies are unexplored.We previously found no correlation or association between the viral loads of the species Iflavirus aladeformis, also known by the virus name deformed wing virus (DWV), and EFB.This observation is not surprising due to the efficient control of the ectoparasitic mites, Varroa spp., which vectors DWV (12)(13)(14).DWV symptoms typically include wing deformities and decreased body size in infected larvae (15), while more commonly subclinical infections have been associated with impaired cognitive function and foraging performance (16)(17)(18).Triatovirus nigereginacellulae, also known as Black queen cell virus (BQCV), causes larval death and decay in queen cells, but infection may be inapparent in worker brood (19,20).Iflavirus sacbroodi, or sacbrood virus (SBV), is often associated with brood that fails to pupate and instead develops in a prepupal shaped sac with a shrunken head and may be inapparent in infected worker bees (21,22).The simultaneous occurrence of brood-rela ted bacterial and viral diseases may exert confounding effects on the overall health of the hive.This complexity is particularly evident in diagnostic processes, as exemplified by the case of Apis cerana bees in India, where the symptoms of Thai sacbrood virus (TSBV) were mistaken for EFB symptoms (23).The symptoms of viral respiratory infections in humans can leave the afflicted individual compromised to secondary bacterial infection, which is associated with severe disease and increased mortality; this is well-recorded with diseases such as pneumonia and influenza (24)(25)(26).Therefore, gaining a deeper understanding of the alterations within the honey bee virome resulting from EFB infection is crucial.This may help elucidate the causal relationship between severe EFB infections and composition changes to the RNA virome as well as determine whether polymicrobial infections play a role in exacerbating disease severity.
The association between DWV and EFB has been briefly investigated in a 2023 study that focused on commercial bees contracted for blueberry pollination, with the authors finding no association between DWV-A or DWV-B variant load and the severity of EFB disease (8).Given that only DWV-A and B variants were monitored for possible associ ation with EFB, our study describes the relationships between EFB and colony based on the whole RNA virome composition.In this study, we used a metagenomic cDNA sequencing approach, as previously described (27,28), using a subset of samples from 12 colonies (N = 6 EFB+, and N = 6 EFB-) taken from two separate yards (yard A and yard B, respectively) that were collected as part of our earlier study (8).This allowed us to explore the viral diversity of the honey bee viromes in EFB symptomatic and healthy colonies and the relationship between EFB disease status and RNA virome composition.

The majority of viral genomes recovered from EFB+ colonies
We generated 6 × 10 8 nanopore sequences across the 12 colonies, assembled into 300 contigs.A total of 41 fragmented and draft viral genomes were binned based on sequence composition and contig coverage with Anvi'o (29,30).Most viral genomes were found in colonies with European foulbrood (EFB+, 34 genomes), while fewer were identified in EFB-colonies (seven genomes).Despite a comparable quantity of reads generated between colonies from both yards and EFB+/EFB-colonies, yard B colonies that were EFB-still had an overall smaller proportion of viral reads taxonomically classified by Kaiju (Table 1; Data File S1).Viral reads overall contributed 0.5%-4.8% of the total cDNA sequenced, while Melisoccocus plutonius, the primary candidate as the causative agent of EFB, contributed between 0.001% and 0.028% of the data set.Notably, M. plutonius transcripts were absent or very low (~0.001%) in EFB-colonies.Between 8% and 18% of the reads were classified as eukaryotic and the majority as unclassified, 70%-87%.This is likely a result of Kaiju being specialized for microbial classification (31), and we confirmed this by mapping the reads to the A. mellifera genome (GCF_003254395.2,accessed Feb 2022), which estimated that the majority of the reads (70%-89%) belong to the host (Table 1; Data File S1).

Lake Sinai virus and sacbrood virus load and diversity distinguish EFB+ and EFB-colonies
Genomes representing LSV were the most abundant (13 genomes, Fig. 4A) and represented five described clades of LSV: two LSV-1 genomes, two LSV-2, three LSV-3, one LSV-4, two LSV-8, and three genomes that did not cluster with any of the pres ently described LSV clades (Fig. 1).Notably, genomes of LSV-1, LSV-4, and LSV-3 were exclusively present in EFB + colonies, whereas LSV-2 genomes were detected in both EFB + and EFB-colonies.LSV-8 genomes were only recovered from yard B colonies that were EFB-and finally LSV-2 and LSV-4 genomes from yard A colonies.This is additionally supported by rank-abundances of viral genomes between the sites, where we observed low-ranked LSV-8 genomes in the yard A colonies and highly ranked in yard B colonies (Fig. 4B).Furthermore, the two LSV-8 variants clustered with a clade within LSV-8 that originated from South Africa and Africanized honey bees in Guayana (27).The origin of Africanized bees in South America was from South Africa, which indicates that these colonies, which originated from Florida, may have been exposed to Africanized bees, which have previously been reported in Southern US states (34,35).There remains a very limited understanding of the impact of LSV on colony health.Still, they are reported as globally widespread (27,36).LSV-2 in particular has been implicated in colony health for migratory bee colonies (37).Overall, LSVs were the most abundant genomes in EFB-colonies, as measured by read-recruitment (Fig. 4).What impact LSV has on EFB disease has not been investigated, but the observations here indicate a phylogenetic distinction between EFB +and EFB-infecting LSV genomes (Fig. 1).
In this data set, we observed during EFB + infection an increase in the fraction of virome reads assigned to DWV and IAPV genomes in EFB + colonies, while BQCV does not differ between EFB + and EFB-colonies.Furthermore, our study noted an increase in read recruitment to binned SBV genomes in EFB + colonies.In prior research, where SBV has not consistently correlated with colony health decline (38), our findings indicate a prevalence of SBV in EFB + colonies (Fig. 4B).The results in this study are impeded  from EFB-colonies, along with additional EFB-, clustered closely together with North American variants (Fig. 3).Our observation might indicate a form of viral dysbiosis in the event of EFB infection, which shifts the community toward one abundant in SBV.This is particularly interesting as EFB is a brood disease like SBV, and exploring the impact on brood viral composition during EFB may reveal more.While SBV can be asymptomatic in adult bees (39)(40)(41)(42), high densities in hives recovering from EFB may be detrimental to the brood and the recovery of the hide during treatment for EFB.

DWV-A and DWV-B are present in EFB+ and EFB-colonies
The data presented here support the original study (Fig. S3), which within the larger data set found no difference in means of the viral loads of DWV between diseased and healthy colonies, as measured by RT-qPCR (8).In this study, we can add the additional support of genomic similarity between DWV genomes recovered from both diseased and healthy colonies.Of the DWV genomes assembled, five represent variant A (DWV-A) and four variant B (DWV-B) (Fig. 2).Only DWV-A genomes were classified as high-quality genomes.Two genomes of low quality clustered with DWV recombinant genomes, and due to short contig length (3.00 and 3.21 Kbp, Data File S2), taxonomy could not be fully ascertained.DWV-A genomes clustered closely with a DWV genome isolated from North American bees (Fig. 2).All DWV-B genomes from the data set clustered closely in the tree and were recovered from yard A colonies, with all but a single genome from a colony with severe EFB symptoms, and the closest neighbors are genomes isolated from European colonies.Notably, in EFB-colonies from yard A, we observed a notable abundance of both DWV-A and DWV-B, a trend not mirrored in colonies from yard B (see Fig. 4B).In EFB-compromised colonies in yard A, there was a decrease in the ranking of DWV-A and DWV-B, with SBV emerging as the predominant genome.Despite DWV-A and DWV-B not being the dominant genomes in yard B colonies, a similar shift in dominance was evident with the onset of EFB.

Dicistroviridae viruses have a limited presence in EFB-colonies
Genomes for members of the viral family Dicistroviridae, which include IAPV and BQCV, were recovered only from EFB +colonies (Fig. S1B).The recovered BQCV genomes were predominantly of medium or low quality, originating from EFB +colonies and showing clustering with a nearby North American BQCV genome (Fig. S1).BQCV was found at relatively low abundances across all samples (Fig. 4A), except for a singular EFB +colony in yard A and an EFB-colony in yard B. However, no complete genomes were obtained from the EFB-colony.Notably, BQCV genomes were exclusively recoverable from EFB +colonies, highlighting its possible association with this brood disease in honey bees.Prior marker gene surveys have shown BQCV as prominent in queen bees (43) and have been associated with colony losses for both A. cerana and A. mellifera in multiple countries (44,45) and globally widespread in bee operations (46)(47)(48).Unfortunately, there is little prior work exploring the interactions of brood-afflicting viruses and EFB.
IAPV is a member of the AKI virus complex, assembled from this data set (49), and ranked in mid to low abundance in all situations, except for EFB +yard B colonies (Fig. 4B), where it ranked highly just below SBV and LSV.A previous marker gene study has shown that bees exposed to migratory management during adulthood had increased levels of the AKI virus complex (50).Although the colonies analyzed in this study were reloca ted from Florida to Michigan for blueberry pollination, the lack of information about the colony's virome composition before transportation limits our ability to assess the potential impacts of the relocation and long-term changes the colonies have undergone as a result of transportation and the onset of EFB symptoms.Future work addressing the correlation between viral infections and EFB may need to consider such variables.

Viral composition varies with colony yards
Despite the constraints posed by the small sample size (n = 12), insights into commun ity composition were gained through an analysis of variance using distance matrices (ADONIS) (Fig. 4C).The results of the ADONIS test indicate that the EFB health status (EFB +or EFB-), yard (A or B), and virus species, as well as their interactions, collectively explain a proportion of the variation observed in the data set (Table S1).Specifically, virus species demonstrate a statistically significant effect (F = 3.709, P = 0.001).Additionally, the interaction between EFB health status and virus species (F = 3.171, P = 0.001) and that of yard and virus species (F = 2.342, P = 0.007) also exhibit significant effects.However, the main effects of EFB status and yard individually show only marginal significance (EFB: F = 2.438, P = 0.058; Yard: F = 1.127,P = 0.302), suggesting that while they may contribute to the model, their impact is not as pronounced as the virus species and its interactions.Overall, the results underscore the importance of consider ing the combined effects of EFB, yard, and virus species, as well as their interactions, in explaining the observed patterns in the data set.These findings, while informative, must be interpreted cautiously due to the inherent limitations of the small sample size and the observed variation between colonies kept in different yards.Furthermore, distinct management approaches to colonies between the two yards may additionally contribute to the variation.The samples utilized in this study were collected during advanced stages of the disease, limiting our understanding of the temporal changes to the virome composition during disease progression.Colonies within the same yard, acting as replicas, exhibit distinct viral compositions.This may influence the study design and underscores the importance of considering this factor, especially when comparing bee colonies held in multiple locations.Understanding whether viral infections worsen EFB severity underscores the importance of early detection and continuous monitoring of the viral consortium throughout.

Conclusion
In this study, we report the RNA viromes from honey bee colonies at two sites from a commercial operation in Michigan.In this study, we observe a distinct community structure in the viromes of colonies kept in two distinct yards, regardless of EFB disease.The original study, from which this data set was subsampled, focused only on DWV and detected no relationship between hygienic behavior and progression of EFB.We observed that SBVs were dominant in EFB +colonies, while LSVs were highly prevalent in EFB-and EFB +colonies, with specific clades associated with EFB +and EFB-colonies.We further observe large variations between colonies from two different yards.This study is limited in scope and size and requires expansion, but it highlights the need for a better understanding of the effects of bacterial brood diseases on the viral dynamics in infected colonies.

Experimental design
The full study design, acquisition, and storage of honey bees can be found in the original publication (8), of which we subsampled 12 colonies, from a commercial beekeeping operation in Southern Michigan for metagenomic analysis.Yard A consisted of colonies (n = 6) used for blueberry pollination, and yard B colonies (n = 6) were kept in a large holding yard away from crop pollination, but in the same region of Michigan (Supp.Data File 1); our subset consisted of three EFB +and three EFB-from each yard.Nurse bees were collected from each colony using a sterile 60-mL centrifuge tube stored at 4°C in the field and transferred to a −20°C environment until processing and transported to the University of Minnesota on dry ice, before being stored at −80°C.European foulbrood disease was confirmed through the culture of Melissococcus plutonius from diseased colonies as previously described (8).

RNA extraction, cDNA synthesis, and nanopore sequencing
As previously described (27,28), pooled honey bees (n = 30) were dissociated with 15 mL of molecular-grade water using gentleMACS M Tubes (Miltenyi Biotec, USA) and a gentleMACS Octo Dissociator (Miltenyi Biotec, USA).The bee homogenate was allowed to settle and 1 mL transferred to a centrifuged tube and centrifuged at 20,000 x g for 10 minutes at 4°C.A 200-uL aliquot was used for RNA extraction using a Nucleo Mag Virus kit (Macherey-Nagel, Düren, Germany) and a magnetic particle processor (MPP) (KingFisher Flex, Thermo Fisher Scientific, USA).Complementary DNA (cDNA) was synthesized.For the first-strand synthesis, 10 uL of viral RNA, 1 uL of N6 Primer II A (24 uM, TakaraBio, USA), 1 uL of SMARTer IIA Oligo (10 uM, TakaraBio, USA), and 1 uL of 10 x Template Switching Reverse Transcriptase (New England Biolabs, MA, USA) were used.A 1 uL volume of Primer IIA (12 uM, TakaraBio, USA) and 1 uL of PrimeSTAR GXL polymerase (TakaraBio, USA) were used for the synthesis of the second strand following the manufacturer's user manual instructions (TakaraBio, USA; New England Biolabs, MA, USA).The synthesized dscDNA was purified using SPRI AMPure beads according to the manufacturer's instructions.cDNA concentrations were quantified by Qubit 4 Fluorome ter (QubitTM4 Fluorometer, Thermo Fisher Scientific, USA) using the dsDNA HS assay kit (Thermo Fisher Scientific, USA).Nanopore sequencing library was prepared with the rapid barcoding sequencing kit (SQK-RBK004) and sequenced on a FLO-MIN106 R9 flow cell for 24 hours.Basecalling was performed using the high-accuracy base-calling model with a minimum Q-score of 9 and a minimum read length of 200 bp.
Contigs with a minimum length of 2 kbp were manually binned with the anvi'o v7.1 interactive interface (29,30).Briefly, anvi'o profiled the contigs using Prodigal v2.6.3 (53), with default parameters, and then reads were mapped to the contig database using Minimap2 v2.24 (54).Read recruitment was stored as a BAM file using Samtools v1.19.2 (55).Anvi'o profiles each BAM file, estimating the read coverage and detection statistics of each contig.The coverage was combined into a merged profile database.The contigs database was populated with additional data, including HMMER search results against virus orthologous groups (VOGs; https://vogdb.org/) in addition to the standard anvi'o HMMR profiles, NCBI COGs, and KEGG KOfam database (56)(57)(58)(59).Contig taxonomy was predicted by running Kaiju v2.9 using the non-redundant NCBI database and RVDB database (accessed 2023) on the gene calls (31,32).Finally, the anvi'o profile was visualized for manual binning.Viral binning was guided by sequence composition similarity (visualized as a dendrogram in the anvi'o interface) and the presence of viral HMM hits to the VOGs.

Validation of draft and fragmented RNA virus genomes from binned contigs
Publicly available honey bee virus genomes were downloaded from the NCBI and deduplicated at 95% average nucleotide identity over 80% contig length using CD-HIT-EST (60,61), retaining the longest genome.Publicly available genomes and genomes generated here were aligned with MAFFT v7.520 (62,63), and IQ-TREE v2.0.3 (64,65) was used for inference of maximum-likelihood trees with 1,000 bootstrapping.Trees were visualized, and metadata were added using the R package ggtree v3.18 (66).

Statistical analysis and data visualization
All data were visualized using R v4.3.0,largely using ggplot2 v3.4.4.Viral genomes were clustered based on 98% nucleotide identity, retaining the longest contig using CD-HIT v4.8.1 (60), generating a non-redundant pool of contigs that represents the viral "population" and removing duplicate viral genomes to estimate viral genome relative abundances (61).Metagenomic reads were mapped to the non-redundant pool of viral genomes using Minimap2, and Samtools was utilized to estimate the number of reads mapping to each contig.An abundance matrix was calculated using the R package MetagenomeSeq v1.43.0 using cumulative sum scaling, a normalization method akin to median-like quantile normalization, to address disparities in sampling depth (67).For multivariate analysis of viral composition, normalized mapped read counts were utilized in conjunction with the vegan package and its metaMDS function.Additionally, an ADONIS analysis, evaluating the impact of yard and EFB symptom status, was performed using the vegan v2.6-7 package (68).

FIG 1
FIG 1Whole-genome maximum-likelihood phylogeny of viral genomes from EFB symptomatic and asymptomatic bee viromes for Lake Sinai virus.Branches are colored by the origin of viral genomes (see Supplementary Data file 1 for full metadata of the genome used in phylogenies).Branch support values are from left to right, and bootstraps from 1,000 replicates are reported as a proportion out of 100.Viral genomes accessed from the NCBI are denoted by their accession numbers, while genomes assembled in this study are annotated with their EFB health record, their holding yard, and genomes by their quality.

FIG 2
FIG 2Whole-genome maximum-likelihood phylogeny of viral genomes from EFB symptomatic and asymptomatic bee viromes for deformed wing virus.Branches are colored by the origin of viral genomes (see Supplementary Data file 1 for full metadata of the genome used in phylogenies).Branch support values are from left to right, and bootstraps from 1,000 replicates are reported as a proportion out of 100.Viral genomes accessed from the NCBI are denoted by their accession numbers, while genomes assembled in this study are annotated with their EFB health record, their holding yard, and genomes by their quality.

FIG 3
FIG 3 Whole-genome maximum-likelihood phylogeny of viral genomes from EFB symptomatic and asymptomatic bee viromes for sacbrood virus.Branches are colored by the origin of viral genomes (see Supplementary Data file 1 for full metadata of the genome used in phylogenies).Branch support values are from left to right, and bootstraps from 1000 replicates are reported as a proportion out of 100.Viral genomes accessed from the NCBI are denoted by their accession numbers, while genomes assembled in this study are annotated with their EFB health record, their holding yard, and genomes by their quality.

FIG 4 (
FIG 4 (A) RNA virome community composition nested by EFB symptoms and colony sites.(B) Rank abundance (mean) of each viral genome (grouped by the lineage and variant).(C) MDMS plots of a Bray-Curtis dissimilarity matrix of cumulative sum scaled normalized read counts to the viral genome collection, colors represent the EFB status, and shape colony yards and datapoint size represent the size of the sequencing library.

TABLE 1
Summary data generation for Oxford Nanopore Technologies sequencing reads by taxonomic classification and viral genome assembly a a Shown here are only reads that met the minimum quality threshold, 200 bp and Q > 9. (EFB, European foulbrood).b Apis-classified reads were estimated by mapping Minimap 2 to the A. mellifera genome due to low success in eukaryotic classification with Kaiju.